The present invention relates to a method for increasing the efficiency of inducing pluripotent stem cells, and more particularly, to a method for increasing the efficiency of inducing pluripotent stem cells by utilizing genes Jhdm1b and Jhdm1a that modify histone.
China is a populous country in the world and also has the highest number of organ losses, damages, failures, and functional disorders as a result of trauma, disease, aging, and heredity. Classical medical therapies based on drugs and surgeries have failed to satisfy the tremendous demand of clinical medicine. As a result, the research on the stem cells and the regenerative medicine attracts the attention of numerous research entities and all sectors of the society.
The cell transplantation therapy constitutes an important research direction of the regenerative medicine, and specific types of cell transplantations may be used to treat heart injury, nervous system degenerative diseases, spinal injury, renal failure, hematological system diseases, and so on. However, the cell transplantation therapy is facing many intricate problems such as allograft rejection and limited cell sources.
The stem cell is a type of cell capable of selfrenewal and can differentiate into various functional cells under certain conditions. Based on their development stage, the stem cells are divided into embryonic stem cells and adult stem cells. Based on their development potential, the stem cells are divided into three types: totipotent stem cells, pluripotent stem cells, and unipotent stem cells. The stem cell is a type of immature cell that is not fully differentiated and has a potential function to regenerate various tissues and organs and the human body, so it is called “universal cell” in the field of medicine.
In order to solve the problems encountered by the cell transplantation therapy, the transformation of cell fate attracts the attention of more and more scientists. Although the determination of cell differentiation and fate has always been considered as an irreversible and stable process in the development process, there are more and more in vitro evidences showing that this process is reversible.
The study on regulation of cell fate is just in a laboratory investigation state and is far from clinical trial. These transformed cells obtained through over-expression of transcription factors still have many application problems, for example, viral sequences integration, potential oncogenicity, the purity of the resultant transdifferentiated cells, and whether they can make up for normal cells which are damaged in certain conditions and play their due roles in the organism.
The induced pluripotent stem cell (iPS) is a type of cell that resembles with embryonic stem cell and has development totipotency. It acquires the properties of the stem cell by inducing the somatic cell through introducing specific ES enriched genes. In 2006, Japanese scientist Yamanaka introduced 24 candidate genes into mouse fibroblasts by using a retrovirus based vector, screened FBX15 positive cells by means of G418 resistance to isolate iPS clones similar to embryonic stem cells, and finally identified that 4 factors including Oct3/4, Sox2, c-Myc, and Klf4 are sufficient to induce mouse FBX15-iPS cells; as compared with embryonic stem cells, these cells are similar with embryonic stem cells in the aspects of clone shape, proliferation capability and ability to form teratoma, but they are different from embryonic stem cells in terms of gene expression and genomic methylation profile and cannot obtain living chimeric mice. Afterwards, this group and other two groups changed the screening method, and they used Nanog as the standard and obtained iPSs that are similar to embryonic stem cells in many aspects and these iPSs can produce chimeric offspring. Recently, the three research groups independently confirmed, by tetraploid complementation test, that mouse iPS cells can develop into an individual and possess development totipotency.
Following the method of inducing mouse iPSs, in 2007, each of the two groups Yamanaka [8] and Yu Junying [9] successfully reprogrammed human somatic cells into iPS cells, wherein the former transduced Oct3/4, Sox2, c-Myc, and Klf4 into human epidermal fibroblasts by using a retrovirus, while the latter incorporated Otc3/4, Sox2, Nanog, and Lin28 into foreskin cells by using a lentivirus. Both of the analysis on gene expression profiling and the analysis on the methylation of the promoter regions of genes Oct3/4 and Nanog showed that the human iPS cell line is very similar to the corresponding embryonic stem cell line, and all of the cells can develop into 3 germinal layers when they are injected into the body of a nude mouse. Furthermore, somatic cells can be successfully induced into iPSs in rat, swine, and monkey, in addition to mouse and human.
The cells that can be successfully reprogrammed are not only limited to fibroblasts, and many other types of adult cells can also be successfully induced into iPS cells, including pancreas beta cells, adult neural stem cells, hepatocytes, gastric cells, mature B cells, haematopoietic cells, meningocytes, adipose-derived stem cells, cord blood cells, peripheral blood CD34 positive cells, and keratinocytes. For cells at different differentiation stages, the difficulties in inducing and reprogramming them into iPSs are different. Take mouse haematopoietic cells as an example: the reprogramming efficiency of haematopoietic stem cells and haematopoietic progenitor cells may be up to 28% which is 300 times that of terminally differentiated T cells and B cells.
In inducing iPSs, it is often to incorporate an exogenous gene into cells by means of a retrovirus and a lentivirus, which provides very high gene transduction efficiency. However, integration of the viral sequence into the genome of the cell may result in gene insertional mutagenesis and even carcinogenicity, so this gene introduction method having potential risks is obviously unfavorable to application of the iPS technique in the field of regenerative medicine. Therefore, a different study group used non-integrating vectors to induce iPSs and succeeded. These vectors include an adenovirus vector, a common expression vector, a transposon, an episome vector, and a minicircle DNA vector.
Both of the combination of Sox2, Klf4, Oct3/4 and c-Myc and the combination of Sox2, Oct3/4, Nanog and Lin28 can successfully induce the generation of iPSs. Further studies found that c-Myc is not essential for reprogramming and the three transcription factors including Sox2, Klf4 and Oct3/4 are sufficient to drive the reprogramming of human and mouse somatic cells. Neural stem cells endogenously express high levels of Sox2, Klf4, and c-Myc, so it only needs to incorporate exogenous Oct3/4 in order to successfully induce iPSs. Among the transcription factors used in reprogramming, Sox2, Klf4, and c-Myc can all be replaced by other members of the same family, for example, Klf2 and Klf5 can replace Klf4; Sox1 and Sox3 can replace Sox2; N-Myc and L-Myc can replace c-Myc; but Oct1 and Oct6 cannot replace Oct4. Esrrb directly binds to Oct3/4 protein to regulate the self-regeneration and totipotency of stem cells, and in reprogramming, Esrrb can replace Klf4 to induce iPSs in combination with Sox 2 and Oct3/4. Oct3/4 is a very important transcription factor in reprogramming. Recent studies found that nuclear receptors LRH-1 (Nr5a2) and Nr5a1 can replace Oct3/4 and can induce mouse adult cells into iPSs in combination with Klf4 and Sox2.
However, so far, there are several different combinations of transcription factors capable of reprogramming, including Oct4, Klf4, Sox2, and c-Myc; Oct4, Nanog, Lin28, Sox2; Sox2, Klf4, and Lrh1; Oct4 and bmil, as well as reprogramming-related genes such as esrrb and tbx3. For the transcription factor combinations required by existing reprogramming methods, it needs to incorporate as many as 3 or 4 transcription factors and the induction efficiency is low. How to reduce the number of transcription factors while maintaining a high reprogramming efficiency is of great importance for reducing the accumulation of cell mutations in reprogramming and for improving the operability of the reprogramming technique. Furthermore, searching for genes that replace common transcription factors facilitates the study of the reprogramming mechanism and the improvement of the reprogramming technique.